Abstract: Bacteria typically live in complex sociomicrobiological communities, often as biofilms, on surfaces as diverse as water pipes, ship hulls, plant roots, insects, shellfish, indwelling medical devices, and mucosal surfaces. Biofilm growth in humans can cause or exacerbate disease, and biofilm growth in environmental niches can facilitate transmission of pathogenic bacteria to humans and other animals. Understanding how bacteria recognize, cooperate and compete with their neighbors in diverse environments is critical for developing strategies to control microbiological community composition, to prevent biofilm development, and to eliminate pre-existing biofilms and their consequent diseases. Contact-Dependent Growth Inhibition (CDI) is a phenomenon in which bacteria use the toxic C-terminus of a large exoprotein to kill or inhibit the growth of neighboring bacteria upon cell-cell contact. Production of a small immunity protein protects bacteria against CDI. Using the Gram-negative bacterium Burkholderia thailandensis as a model, we have discovered that in addition to using CDI system proteins to kill their neighbors, bacteria can use these proteins for signal transduction, causing a change in gene expression that leads to the production of cooperative behaviors, such as biofilm formation, when neighboring bacteria are recognized as ?self?. Here, we propose experiments to determine the molecular mechanisms underlying this Contact-Dependent Signaling (CDS) phenomenon. Our preliminary data support a model in which the C-terminus of the large BcpA exoprotein is delivered into neighboring bacteria where it forms a ?CDS complex? that catalyzes a reaction that increases or decreases the concentration of a signal molecule, which may be a small nucleotide second messenger. Experiments described in Aim 1 will define the CDS regulatory network and identify the signal molecule, which will help us identify the enzymatic activity of the CDS complex. Experiments described in Aim 2 will identify the members of the CDS complex. We will reconstitute the CDS complex in vitro using purified proteins and will measure its catalytic activity. We have discovered that a small protein called BcpO is required for CDS. We proposed experiments in Aim 3 to determine if BcpO localizes to the inner leaflet of the outer membrane, as predicted, and if that localization is important for its function. We will also identify proteins with which BcpO interacts so that we can understand how it functions in CDS.